Kinetoplastid protozoa contain a unique microbody organelle called the glycosome. Several important metabolic pathways are compartmentalized within the glycosome that are found in the cytoplasm of higher eukaryotes. We have previously reported the identification of a Leishmania donovani cell line called gim1-1, in which several normally glycosomal proteins are partially mislocalized to the cytoplasm. The GIM1 gene complements the defect and restores import of proteins to the glycosome. Here we demonstrate that GIM1 encodes an integral membrane protein of the glycosome. We also report that the mutant gim1-1 allele behaves as a dominant negative mutation. Introducing the gim1-1 allele extrachromasomally led to mislocalization of a glycosomal reporter protein even in wild-type cells. Gene disruption experiments in heterozygous GIM1/gim1-1 cells showed that when the mutant gim1-1 allele was lost, cells re-established normal glycosomal protein localization. Interestingly, no disruptions of the wild-type allele were obtained. These data indicate that a dominant negative mutation in the GIM1 gene is the sole genetic lesion responsible for the glycosomal defects in gim1-1, and suggest that GIM1 is an essential gene in Leishmania.